Rett Syndrome

Presented by: Ashley Owen

University of Wisconsin-Eau Claire

November 30, 2004

What is RTT?

Neurodevelopmental disorder cased by mutations in the methyl-CpG-binding protein 2 (MECP2) and is characterized by the loss of acquired skills after a period of normal development in infant girls.

Symptomsafter 7 to 18 months

Mild learning difficulties Disturbances with

breathing and cardiac rate

Bowel immobility Screaming fits Autistic features Microcephaly Seizures

Hand stereotypes Washing, clapping,

mouthing

Decrease in head growth

Small statue Teeth grinding “eye pointing”

Inheritance

Prevalence 1/10,000-15,000 females

X-linked dominant mutation De novo Inherited from parent with the disease causing

mutation and germline mosaicismMother would have XCI and be unaffected

Males

47, XXY Identified as RTT

Somatic mosaicism XCI

46, XY Severe neonatal encephalopathy

Leads to death“Disease of the brain”

Gene of Interest: MECP2

Methyl-CpG-Binding Protein Two domains

Methyl-CpG-binding domain (MBD) Transcriptional repression domain (TRD)

Location: Xq28 Pericentromeric heterochromatin

Transcriptional silencing/repression, epigenetic regulation, nuclear structure (chromatin) 5-methylcytosine rich heterochromatin

MBDNan, X., Meehan, R.R., & Bird, A.

Located between amino acid 89 and 162

Symmetrical methylated CpG dinucleotides

Binds to minor groove of beta DNA

MBD(cont.) Nan, X., Meehan, R.R., & Bird, A.

Dimerization is not required for binding Monomer

MBD and TRD are important for XCI Methyl dependent

repression

TRD

Interacts with co-repressor Sin3A Recruits histone deactylases

Exons

Exon 1 Non-coding 5‘

untranslated region (UTR)

Exon 2 Coding sequence

Exon 3 Coding sequence

Exon 4 Non-coding 3‘ UTR Coding sequence Polyadenylation creates

different protein lengths

Structure of Human MECP2Wan, M., et.al.

DetectionLewis, J.D., et.al.

Looking for MECPs expression clone

Methylated and unmethylated probe

Differ from MECP1 MECP1 is a 120kb Tissue distribution

Testis Anion/cation ion exchange

column Binding specificities

12 methyl-CpGs vs. a pair

Obtaining cDNALewis, J.D., et.al.

Partial amino acid sequence as a primer

340 bp fragment from original PCR to complete library λZAPll

ORF 492 amino acids and all 6 major peptides

Translational Experiments Lewis, J.D., et.al.

SDS-polyacrylamide Relationship between

ORF and MECP2 Showed an 81kd

sequence cDNA fused into E.Coli

with ß-galactosidase gene

Bound to methylated probe

Conclusion: ORF codes for MECP2

Localization Lewis, J.D., et.al.

Immunofluorescence Ab76 serum

Stained in heterochromatin areas

Parallel satellite DNA in mice Contains 8 CpGs sights

Associated with pericentromeric heterochromatin

Comparison of Mouse and Human MECP2

Reichwald, K., et.al.

•Identity between the mouse and human gene is 68% (average)

Mechanism

Expressed during organogenesis during embryonic life and in the hippocampus during adult life Other methylated binding proteins take over in

other cells during adulthood

Believed to be involved in XCI and genetic imprinting

Tissue Specific

Two transcripts 1.9kb ~10kb

Difference in tissue expression Difference in

translatability Half life is similar

Mutations

99.5% are sporadic Nonsense, missense/frameshifts, deletions

Majority are nonsense Detected using PCR and restriction enzyme

analysis Occur in CpG sites

HypermutableMethylated in germline and prone to deamination (C to T)

Mutations(Cont.)Wan, M., et.al.

See word document

Structure of Human MECP2Wan, M., et.al.

TreatmentNo trxt has shown significant improvements

Previous trxts L-carnitine

Fatty acid metabolism Respiratory features

Ketogenic diet Control of seizures Lacked vitamins

Naltrexone* Oral opiate antagonist Respiratory features,

EEG patterns

Ongoing trial Folate-Betaine

Methyl-donor group Alter gene expression Recruit other methyl

binding groups

Current trxt Supportive/symptomatic

therapy Occupational/physical

therapy

Social and Ethical Dilemmas

Money…testing in general is expensive Have to show clinical signs/family member Diagnostic testing

Test for mutation prenatallyPossible involvement with other disorders

Mental retardation in males

References

Hagberg, B.A. and Skjeldal, O.H. (1994). Rett variants: A suggested model for inclusion criteria. Pediatric Neurology, 11, 5-11.

Lewis, J.D., et.al. (1992). Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell, 69, 905-914.

Nan, X., Meehan, R.R., & Bird, A. (1993). Dissection of the methyl-CpG binding domain from the chromosomal protein MeCP2. Nucleic Acids Research, 21, 4886-4892.

Percy, A. K. (2002). Clinical trials and treatment prospects. Mental Retardation and Developmental Disabilities Research Reviews, 8, 106-111.

Reichwald, K., et.al. (2000). Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions. Mammalian Genome, 11, 182-190.

Rett Syndrome, RTT. (2004). Online mendelian inheritance in man, 9 Oct. 2004. Shahbazian, M.D., Antalffy, B., Armstrong, D.L. & Zoghbi, H.Y. (2002). Insight into rett

syndrome: MECP2 levels display tissue- and cell-specific difference and correlate with neuronal maturation. Human Molecular Genetics, 11, 115-124.

Wan, M. et.al. (1999). Rett syndrome and beyond: Recurrent spontaneous and familial MECP2 mutations at CpG hotspots. American Journal of Human Genetics, 65, 1520-1529.

Zoghbi, H.Y. (2004). Rett Syndrome. GeneReviews, www.genetests.org, 29 Sept. 2004.

Questions